Fig. 2.

Evaluation of TMR-Noxa peptide in dual-readout F² assay in 1,536-well format. Increasing concentrations of TMR-Noxa peptide were incubated with 50 nM Mcl-1 protein at room temperature for 1 h. The total reaction volume is 5 (L per well. (A) Total FI values were measured for each TMR-Noxa peptide concentration and compared to those of buffer alone in the presence or absence of Mcl-1 protein. (B) The polarization signal was recorded and expressed as FP signal windows after subtracting the mP values for tracer alone. The data shown are average values with SD from four replicates. (C) TR-FRET signal with increasing concentrations of Noxa-Rho peptide were measured and plotted against TMR-Noxa peptide concentrations. TR-FRET signal = A_572nm/A_545nm × 10⁴; (D) the S/B values of TR-FRET (expressed as the calculated values × 2 to approximate similar scale) or S/N values of FP were obtained from data in (B) and (C) and plotted against TMR-Noxa peptide concentrations. (E) Z′ factors of the assay were calculated for both TR-FRET and FP measurements from the data in (B) and (C). FI, fluorescence intensity; mP, millipolarization; S/B, signal-to-background ratio; SD, standard deviation; S/N, signal-to-noise ratio.